Milk is one of the most thoroughly researched foods in history. Countless scientific papers document the composition of milk and describe the biological functionalities in this complex bio-resource. Proteins, peptides, enzymes and other biomolecular substances constitute major and very important component of milk and are believed to be responsible for many of the specific functionalities passed on from a mother to her new-born in addition to basic nutrients.
Casein is the colloidal protein constituent of milk which can be precipitated e.g. by treatment with rennet enzymes under neutral or weakly acid pH conditions or by means of a mineral salt, such as a calcium salt, or by means of acids at a pH of e.g. from 4.6 to 5.6.
Casein in milk is precipitated during a number of different dairy-related industrial processes, e.g. cheese production and casein or caseinate production.
During cheese manufacturing, the casein is precipitated by e.g. a rennet enzyme activity and the casein coagulum (cheese curd) contains entrained but soluble whey proteins, fat, lactose and minerals from milk too.
Cheese whey is a relatively dilute protein solution comprising lactose, minerals and lipids that is obtained as a by-product when producing any type of cheese by the aid of rennet enzymes and/or microbiological starter cultures.
In the past two decades, there has been a significant focus on utilisation of whey proteins e.g. bovine whey proteins from milk. Today, several bovine Whey Protein Concentrates (WPC) and bovine Whey Protein Isolates (WPI) are standard products obtained through various membrane filtration techniques as well as ion exchange adsorption procedures.
Further utilisation of the bovine whey in terms of fractionation of the proteins into individual protein fractions, such as β-lactoglobulin, α-lactalbumin, immunoglobullns, lactoperoxidase, and lactoferrin, is made possible through chromatographic packed bed and expanded bed separation techniques. Protein products from chromatographic separation technologies are generally characterised by their low- to non-fat content and are useful for a broad range of applications e.g. within foods, feeds, functional foods, and health care products.
Since the first market introductions of WPC and WPI products and more recently, the first purified single protein products comprising lactoferrin, an ever increasing demand for even more sophisticated and still more efficient and cost effective production processes has evolved.
Lactoferrin is a metallo-protein found naturally within biological fluids, such as milk and saliva, at mucosal surfaces and within white blood cells at relatively low concentrations. Besides working as a transmitter molecule in iron assimilation for the infant, lactoferrin has an antibacterial functionality towards iron-dependent microorganisms.
Due to its antibacterial, anti inflammatory and immune modulating properties, lactoferrin offers promising new applications, for example in the treatment of diseases, commonly difficult to treat with antibiotics.
Thus a broad range of activities and applications of products comprising lactoferrin have been suggested:                Natural antibiotic activity—killing many disease causing bacteria, whilst protecting the natural bacterial flora of the body.        Immune-boosting activity—lactoferrin aids in maturation and regulation of a number of immune competent cells throughout the body.        Antioxidant activity—the strong binding of iron by lactoferrin, prevents “free iron” from forming free radicals. Free radicals have been implicated as a causative agent in many diseases including cancers.        Antifungal activity—Lactoferrin kills a range of fungi and yeast, including the causative agent of thrush, Candida albicans.        Research in antiviral agents has shown that lactoferrin can prevent viruses, such as HIV, hepatitis and CMV, from binding to the cells of the body and thus preventing viral infection.        Antitumour activity—lactoferrin and related peptides have been shown to suppress tumour growth and prevent tumour formation in animal models.        Iron uptake—As lactoferrin binds very tightly to iron, lactoferrin is believed to allow efficient uptake of iron into the body. This is considered as being beneficial for anaemic patients.        
Furthermore, treatment of meat surfaces with lactoferrin solutions to inhibit bacterial growth and thereby minimise the risk of food contamination and prolong shelf life of certain food products has been suggested as an important future application of lactoferrin.
The concentration of lactoferrin in bovine skimmed milk is usually small, typically between 80-200 mg/ml depending on e.g. the pasteurisation and other pre-treatment history of the skimmed milk. After precipitation of the casein present in milk, the concentration of lactoferrin in bovine whey is typically between 10-100 mg per litre depending on the physical and chemical pre-treatment of the whey.
Thus, there is a need for an effective, simple and cost effective large scale process to enable an industrial production of lactoferrin products to be used in various applications.
During manufacturing of certain cheese products the cheese curd is washed with a salt solution or solid salt is added to the cheese curd which leads to the separation of a protein containing extract. The addition of salt or saturated salt solution to the cheese curd results in syneresis of the curd causing expulsion of water, whey components (fat, protein, lactose, etc) and some salt.
Regarding the nature of such an extract, U.S. Pat. No. 5,783,237 discloses such an extract that is discharged even though it contains valuable milk and whey components since it cannot be usefully reprocessed because of the high salt content and it is difficult to dispose of because of its high Biological Oxygen Demand (BOD) and salinity level.
Thus, the extract is at many production sites regarded as a waste product that has little or no value and in most instances it is a problem to discard the product. Furthermore, it seems to be a general observation that the extract cannot be further processed to take advantage of any valuable compounds present therein due to the high salt concentration.
Casein and caseinates are generally produced from skimmed milk. It is precipitated by treating milk with a destabilising agent, such as an acid, a mineral salt or rennet and then separated from the residual whey by means of a separator or filter. It is then typically washed with large quantities of water and dried.
U.S. Pat. No. 4,976,865 discloses a method of separating biological macromolecules by using packed bed chromatography as well as expanded bed chromatography which is economically feasible to provide an industrial scale production. The compounds are primarily obtained from natural products such as milk. In particular, the method disclosed may be applied to the chromatographic separation of whey proteins, specifically are β-lactoglobulin and α-lactalbumin are extracted from a “mild” whey i.e. whey without increased salt concentration or ionic strength. For elution of β-lactoglobulin and α-lactalbumin a 0.1N hydrochloric acid solution is used. The method does not disclose isolation and purification of lactoferrin. Furthermore, the whey used for isolation and purification of whey proteins is a mild whey and the use of an extract obtained from the treatment of precipitated casein with a salt or a salt solution has not been disclosed.
U.S. Pat. No. 4,791,193 discloses a process for producing lactoferrrin with a high purity from a bovine source. It is disclosed that in spite of the useful physiological functions of lactoferrin, it has been difficult to isolate and purify lactoferrin from milk at an industrial scale because of its minor proportion in milk. The milk used in this invention is in the form of either skimmed milk or as whey. The patent discloses a process utilising a solution of one or more salts selected from the group consisting of sodium chloride, potasium chloride, calcium chloride and magnesium chloride for eluting the whey protein, lactoferrin, captured by a weakly acidic cation-exchanger. The concentrations of the salt solutions applied for elution are in the range of 0.4-2.5 and 1.5-12. U.S. Pat. No. 4,791,193 does not disclose use of an extract for isolation of whey proteins such as lactoferrin. Additionally, the process does not disclose the preparation of an extract obtained from the precipitated milk casein for the refinement of lactoferrin.
U.S. Pat. No. 5,596,082 discloses a process for isolating lactoferrin and lactoperoxidase from milk and milk products and products obtained by such a process. In particular, a process is disclosed for isolating lactoferrin and lactoperoxidase from milk at an industrial scale. The starting material used is whey from cheese manufacturing or neutralised casein whey at pH 6.5 obtained from skimmed milk after casein precipitation. Lactoferrin and lactoperoxidase are adsorbed to a cation exchanger at a high surface velocity and subsequently eluted from the cation exchanger with a salt solution. This patent does not disclose the use of an extract for refining whey proteins such as lactoferrin. Additionally, the process does not disclose the preparation of an extract obtained from the precipitated milk casein for the isolation of lactoferrin.
The above-mentioned processes are based on known chromatographic techniques wherein a lactoferrin-containing liquid passes a solid phase designed to capture lactoferrin by a selective binding.
A disadvantage of the known production processes for lactoferrin is the relatively low concentration of the lactoferrin naturally present in milk and whey raw materials. This implies that large volumes of liquid must be treated in order to produce a certain amount of lactoferrin. The relatively high concentration of other milk/whey proteins makes it challenging and relatively expensive to obtain a product with a high degree of lactoferrin purity.
Furthermore, the preparation of an extract obtained from the precipitated milk casein for the isolation of lactoferrin has not been disclosed in the prior art.
The inventors of the present invention surprisingly found that treatment of the precipitated casein, from e.g. cheese manufacturing, may result in an extract containing an elevated concentration of lactoferrin relative to traditional whey obtained as a waste product in e.g. cheese manufacturing. This lactoferrin-enriched extract is suitable for an efficient recovery of lactoferrin or alternatively, directly applicable in low cost applications on wet or dry basis.
Thus, by providing a process according to the present invention it has become possible to recover whey proteins, such as lactoferrin, in a manner which is cheap, specific, more efficient, easily performed and the invention is easily compatible with automated and semi-automated systems.